Abstract
Nonsteroidal anti-inflammatory drugs (NSAIDs) are used extensively as therapeutic agents, despite their well documented gastrointestinal (GI) toxicity. At this time, the mechanisms responsible for NSAID-associated GI damage are incompletely understood. In this study, we used microarray analysis to generate a novel hypothesis about cellular mechanisms that underlie the GI toxicity of NSAIDs. Monolayers of intestinal epithelial cells (IEC-6) were treated with NSAIDs that either exhibit (indomethacin, NS-398 [N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide]) or lack (SC-560 [5-(4-chlorphenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole]) inhibitory effects on IEC-6 migration. Bioinformatic analysis of array data identified the calpain cysteine proteases and their endogenous inhibitor calpastatin as potential targets of NSAIDs shown previously to retard IEC-6 migration. Accordingly, quantitative real-time reverse transcription polymerase chain reaction and immunoblotting were performed to assess the effects of NSAIDs on the expression of mRNA and protein for calpain 8, calpain 2, calpain 1, and calpastatin. In treated IEC-6 monolayers, NS-398 decreased the expression of mRNA for calpain 2 and calpain 8. Both NS-398 and indomethacin decreased the protein expression of calpains 8, 2, and 1. None of the NSAIDs affected expression of calpastatin mRNA or protein. The calpain inhibitors, N-acetyl-Leu-Leu-methioninal and N-acetyl-Leu-Leu-Nle-CHO, retarded IEC-6 cell migration in a concentration-dependant fashion, and these inhibitory effects were additive with those of indomethacin and NS-398. Our experimental results suggest that the altered expression of calpain proteins may contribute to the adverse effects of NSAIDs on intestinal epithelial restitution.
Footnotes
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This study was supported by the National Institutes of Health (Grant 1P02RR027686), the associated Molecular Biology Support Core, and the Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University. K.W.'s summer research experience was partially funded by the Graduate School at Kansas State University. The Kansas University Medical Center-Microarray Facility is supported by the Kansas University-School of Medicine, the Kansas University Medical Center Biotechnology Support Facility, the Kansas University Medical Center Mental Retardation Research Center (Grant HD02528), and the Kansas IDeA Network of Biomedical Research Excellence (Grant RR016475).
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N.N.R. and K.S. contributed equally to this work.
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Parts of this work were presented at the following meeting: Lillich JD, Silver K, Raveendran NN, and Freeman LC (2007) Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit IEC-6 cell migration and calpain protein expression. 2007 Experiment Biology Meeting; 2007 Apr 20–May 2; Washington, DC. Federation of American Societies for Experimental Biology, Bethesda, MD.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.127720.
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ABBREVIATIONS: NSAID, nonsteroidal anti-inflammatory drug; GI, gastrointestinal; COX, cyclooxygenase; PG, prostaglandin; IEC, intestinal epithelial cell; SC-560, 5-(4-chlorphenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole; NS-398, N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide; DMSO, dimethyl sulfoxide; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; SLR, signal log ratio; IPA, Ingenuity Pathway Analysis; Ct, threshold cycle; ALLN, N-acetyl-Leu-Leu-Nle-CHO; ALLM, N-acetyl-Leu-Leu-methioninal; Indo, indomethacin.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received June 26, 2007.
- Accepted February 14, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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